Diphtheria toxin is a 58,000 dalton protein. The crystal structure of the toxin reveals three distinct domains corresponding to the three functional activities of the toxin: receptor binding, membrane translocation and enzymatic activity. Our studies are directed at a better understanding of the structure-function relationships of the translocation and enzymatic domains. Residues within the enzymatic cleft have been mutagenized to better define to toxin's ability to transfer ADP-ribose from NAD to elongation factor 2. The translocation domain of the toxin reveals a high concentration of a-helical regions. Two the these a-helices are hidden in the center for the domain at neutral pH. It is believed that when the toxin encounters acidic conditions in the endosome, the translocation domain undergoes a conformational change, exposing these two a-helices and allowing them to insert into the lipid bilayer as the initial step in membrane translocation. A proline residue at position 345 is one of the amino acids separating these two a-helices. We have changed this Pro to Glu or Gly and found that while the mutant toxins demonstrated comparable binding activity and enzymatic activity to the native protein, they were 100-fold less toxic to cells reflecting a 100-fold reduction in translocation activity. These results demonstrate the critical role of Pro 345 in membrane translocation.